The present invention lies in the field of immunoassays, specifically providing an immunoassay for quantitative and qualitative measurement of plant pollen tube growth.
The measurement of in-vitro pollen germination and pollen tube growth has been shown to be a sensitive means for measuring the response of plant tissue to a variety of chemical and environmental stresses. For example, the measurement of pollen tube growth has been used to select plants for resistance to herbicides and heavy metal ions. See, for example, Smith, G.A. and Moser, H.S. (1985) Theor. Appl. Genet. 71:231; Feder, W.A. (1986) in Biotechnology and Ecology of Pollen, D.L. Mulcahy, G. V. Mulcahy, E. Ottaviano (Eds.). Springer-Verlag, New York, p. 89; Searcy, K.B. and Mulcahy, D.L. (1985) Amer. J. Bot. 72:1700; Searcy, K.B. and Mulcahy, D.L. (1986) in Biotechnology and Ecology of Pollen (supra.) p 159. The bioassay of pollen tube growth has also been used to investigate the effects on pollen growth of style extracts and glycoproteins associated with self-incompatibility. See Shivanna, K.R. et al. (1981) Protoplasma 107:319; Dickinson, H.G. et al. (1982) Proc. R. Soc. B 215:45; Williams, E.G. et al. (1982) Planta 156:517.
The growth of pollen in-vitro has usually been determined directly, counting the percentage of grains that germinate and measuring pollen tube length by direct measurement. Such methods are tedious, time consuming and wholly unsuited for large samples or for automation. Furthermore, measurements of pollen tube length, whether made using a microscope fitted with an eye piece graticule or a semi-automated image analysis system are, to a certain extent subjective. The present invention provides a new method for measuring pollen tube growth based upon an immunoassay which has been discovered to provide a quantitative measurement of growth.
Chemical studies of the cell wall composition of pollen tubes have been reviewed by Harris, P.J. et al. (1984) Oxford Surveys Plant Molec. and Cell Biol. 1:161-203. Such studies have included the monocotyledons, Lilium, and Tulipa as well as the dicotyledons, Camellia, Petunia, and Nicotiana. Most of the prior studies have been confined to analyses of monosaccharides in acid hydrolysates of whole cell walls or cell wall fractions. The composition of the pollen cell walls of Nicotiana alata have been found to consist primarily of callose, a (1&gt;3)-.beta.-D-glucan and a (1&gt;5)-.alpha.-L-Arabinan as primary components, together with small amounts of cellulose and a uronic acid-containing polysaccharide localized at the pollen tube tip. Rae, A.L. et al. (1985) Planta 166:128.
Although it is sometimes difficult to generate an antibody response to polysaccharides, especially those of animal origin, it is sometimes observed that polysaccharides of plant origin do generate an antibody response. Anderson, M.A. et al. (1984) Plant Physiol. 75:1013, have shown that mice can be immunized with extracts of the styles of mature flowers of Nicotiana alata. The authors reported that about half the hybridomas derived from such an immunization secreted antibodies to N. alata style arabinogalactan protein (AGP). The AGP of N. alatastyles contains 68% carbohydrate of which arabinose and galactose are the major components. Furthermore, a high proportion of the hybridomas were directed to both L-arabinose and D-galactose, since antibody binding to the isolated AGP was inhibited by either L-arabinose or D-galactose, but not by glucose. Other hybridomas were produced which secreted antibody with preference for L-arabinose compared with D-galactose, or with preference for D-galactose compared with L-arabinose. It was found in the studies that a monoclonal antibody could be selected which had preferential binding for L-arabinose residues and that polysaccharides with a single terminal arabinofuranosyl residue were also bound by the antibody. Harris et al. (1984) disclosed that an o-L-arabinofuranosyl directed monoclonal antibody also bound to the surface of in-vitro grown N. alata pollen tubes.
Antibodies, both polyclonal and monoclonal, are wellknown in the art. Given source materials specified as to composition or method of preparation to be used as antigens, a variety of techniques of immunization, purification and selection are well-known in the art for producing polyclonal and monoclonal antibodies. Techniques for preparing monoclonal antibodies (MABs) were initially described by Kohler, G. and Milstein, C. (1975) Nature 256: 495, and more recent publications on the subject include Reuveney, S. et al. (1985) Develop. Biol. Standard 60:185; Bodeus, N. et al. (1985) J. Immunol. Meth. 79:1; and The Commercial Production of Monoclonal Antibodies, S. Seaver (Ed.) Marcel Dekker, Inc., New York (1986). The method used for preparing the monoclonal antibody exemplified herein was described by Anderson, M.A. (1984), supra.
Immunoassay techniques have also been described in great detail in publications over the last 10 to 15 years. Enzyme-linked immunoassays (ELISA) have been described for example by Engvall, E. in Methods in Enzymology 70:419.